A two-protein strategy for the functional loading of a cellular replicative DNA helicase

The delivery of a ring-shaped hexameric helicase onto DNA is a fundamental step of DNA replication, conserved in all cellular organisms. We report the biochemical characterization of the bacterial hexameric replicative helicase DnaC of Bacillus subtilis with that of the two replication initiation proteins DnaI and DnaB. We show that DnaI and DnaB interact physically and functionally with the DnaC helicase and mediate its functional delivery onto DNA. Thus, DnaB and DnaI form a pair of helicase loaders, revealing a two-protein strategy for the loading of a replicative helicase. We also present evidence that the DnaC helicase loading mechanism appears to be of the ring-assembly type, proceeding through the recruitment of DnaC monomers and their hexamerization around single-stranded DNA by the coordinated action of DnaI and DnaB.     

Development of an attractant for the scarab pest Macrodactylus subspinosus (Coleoptera: Scarabaeidae)

Field trials were conducted over several seasons to determine the attractant most successful in luring adult rose chafers, Macrodactylus subspinosus (F.), to traps. During the first season, 20 compounds were compared with the standard lure, valeric acid + hexanoic acid + octyl butyrate (1:1:1). The two new standards establishedthat season were: valeric acid + 1-nonanol (1:1); and valeric acid + hexanoic acid + octyl butyrate + 1-nonanol (1:1:1:1). The following season, 36 compounds were evaluated, comparing them to the new standards. The performance of the standard binary lure valeric acid + 1-nonanol was improved when the alcohol 1-nonanol was replaced by its analog trans-2-nonenol and this was confirmed during the third season. At the same time, a second test was conducted with 29 new candidates, which were combined with valeric acid and compared with the standard: valeric acid + hexanoic acid + octyl butyrate + 1-nonanol (1:1:1:1). A control and the single compound alpha-ionone were included, resulting in the discovery of a new more powerful attractant, alpha-ionone. Testing of alpha-ionone continued the following season, at which time the initial leading candidate and new ones containing trans-2-nonenol were tested against the single attractant alpha-ionone and various combinations of it. A new five-component mixture of valeric acid, hexanoic acid, octyl butyrate, trans-2-nonenol, and alpha-ionone out performed all other lure combinations.     

Microtubule organization and the effects of GFP-tubulin expression in dictyostelium discoideum

We have labeled microtubules in living Dictyostelium amoebae by incorporation of a GFP-alpha-tubulin fusion protein. The GFP-alpha-tubulin incorporates into microtubules and, as reported by others [Neujahr et al., 1998], the labeled microtubules are highly motile. Electron microscopy (EM) analysis of the distribution and organization of microtubules in the amoebae shows that some cytoplasmic microtubules form close associations. These associations could allow motor proteins attached to one microtubule to walk along an adjacent microtubule and thus generate some of the observed motility. Protein blot analysis indicates that the GFP-alpha-tubulin incorporates into microtubules at a lower efficiency than does the endogenous alpha-tubulin. EM and immunofluorescence (IF) analyses suggest that the GFP-alpha-tubulin interferes with microtubule nucleation. We have also observed an increased sensitivity of the GFP-alpha-tubulin expressing cells to blue light, as compared to wild-type cells. These results suggest that although GFP-alpha-tubulin can be used as a marker for microtubules in living cells, the use of this marker is not recommended for certain types of studies such as assembly dynamics.     

LTD₄ induces HB-EGF-dependent CXCL8 release through EGFR activation in human bronchial epithelial cells

Airway epithelial cells release proinflammatory mediators that may contribute to airway remodeling and leukocyte recruitment. We explored the hypothesis that leukotriene D? (LTD?) may trigger the release of proremodeling factors through activation of the EGF receptor (EGFR). We particularly focused on the effects of LTD? on release of heparin-binding EGF-like factor (HB-EGF) and IL-8 (CXCL8), a potent neutrophil chemoattractant that may be released downstream of EGFR activation. To address this hypothesis, both primary (NHBE) and transformed bronchial human epithelial cells (BEAS-2B) were grown on an air-liquid interface and stimulated with LTD?. HB-EGF and CXCL8 were evaluated by ELISA in cell culture supernatants. To explore the EGFR signaling pathway, we used a broad-spectrum matrix metalloproteinase (MMP) inhibitor, GM-6001, two selective EGFR tyrosine kinase inhibitors, AG-1478 and PD-153035, an HB-EGF neutralizing antibody, and a specific small interfering RNA (siRNA) against the EGFR. Expression of the CysLT? cysteinyl leukotriene receptor was demonstrated by RT-PCR and immunocytochemistry in both BEAS-2B and NHBE cells. Four hours after stimulation with LTD?, HB-EGF and CXCL8 were significantly increased in cell culture supernatant. GM-6001 and montelukast, a specific CysLT? receptor antagonist, blocked the LTD?-induced increase in HB-EGF. All inhibitors/antagonists decreased LTD?-induced CXCL8 release. siRNA against EGFR abrogated CXCL8 release following stimulation with LTD? and exogenous HB-EGF. These findings suggest LTD? induced EGFR transactivation through the release of HB-EGF in human bronchial epithelial cells with downstream release of CXCL8. These effects may contribute to epithelial-mediated airway remodeling in asthma and other conditions associated with cysteinyl leukotriene release.     

Divergence genetics analysis reveals historical population genetic processes leading to contrasting phylogeographic patterns in co-distributed species

Coalescent samplers are computational time machines for inferring the historical demographic genetic processes that have given rise to observable patterns of spatial genetic variation among contemporary populations. We have used traditional characterizations of population structure and coalescent‐based inferences about demographic processes to reconstruct the population histories of two co‐distributed marine species, the frilled dog whelk, Nucella lamellosa, and the bat star, Patiria miniata. Analyses of population structure were consistent with previous work in both species except that additional samples of N. lamellosa showed a larger regional genetic break on Vancouver Island (VI) rather than between the southern Alexander Archipelago as in P. miniata. Our understanding of the causes, rather than just the patterns, of spatial genetic variation was dramatically improved by coalescent analyses that emphasized variation in population divergence times. Overall, gene flow was greater in bat stars (planktonic development) than snails (benthic development) but spatially homogeneous within species. In both species, these large phylogeographic breaks corresponded to relatively ancient divergence times between populations rather than regionally restricted gene flow. Although only N. lamellosa shows a large break on VI, population separation times on VI are congruent between species, suggesting a similar response to late Pleistocene ice sheet expansion. The absence of a phylogeographic break in P. miniata on VI can be attributed to greater gene flow and larger effective population size in this species. Such insights put the relative significance of gene flow into a more comprehensive historical biogeographic context and have important implications for conservation and landscape genetic studies that emphasize the role of contemporary gene flow and connectivity in shaping patterns of population differentiation.     

556例多发伤患者的急诊救治临床分析

目的探讨多发伤患者的救治策略。方法回顾分析我科2000年1月至2008年5月急诊抢救的556例多发伤患者的临床资料。结果 16例患者经抢救无效死亡,死亡率2.88%;其余患者均经紧急抢救及行必要实验室检查,病情稳定,好转率达97.12%。平均抢救时间为(1.37±1.05)h。结论强化多发伤的急诊科早期救治,树立创伤急救"黄金1 h"观念,是提高多发伤患者生存率及降低死亡率的关键。     

A key presynaptic role in transformation for a widespread bacterial protein: DprA conveys incoming ssDNA to RecA

Natural transformation is a mechanism for genetic exchange in many bacterial genera. It proceeds through the uptake of exogenous DNA and subsequent homology-dependent integration into the genome. In Streptococcus pneumoniae, this integration requires the ubiquitous recombinase, RecA, and DprA, a protein of unknown function widely conserved in bacteria. To unravel the role of DprA, we have studied the properties of the purified S. pneumoniae protein and its Bacillus subtilis ortholog (Smf). We report that DprA and Smf bind cooperatively to single-stranded DNA (ssDNA) and that these proteins both self-interact and interact with RecA. We demonstrate that DprA-RecA-ssDNA filaments are produced and that these filaments catalyze the homology-dependent formation of joint molecules. Finally, we show that while the Escherichia coli ssDNA-binding protein SSB limits access of RecA to ssDNA, DprA lowers this barrier. We propose that DprA is a new member of the recombination-mediator protein family, dedicated to natural bacterial transformation.     

The Role of Climate in Settlement and Landscape Change in the North Atlantic Islands: An Assessment of Cumulative Deviations in High-Resolution Proxy Climate Records

In order to assess possible contributions of climate change to the human ecology of the North Atlantic islands we evaluate the utility of cumulative deviations from the mean, calculated for the Greenland ice core storm frequency proxy (GISP2 Na⁺) and sea ice proxy (GISP2 chloride excess). Our aim is to identify episodes of unpredictable change in the context of long-term trends of cultural and environmental development. Key changes are identified in the proxy climate records in 975 and 980 ad, 1025 and 1040 ad, 1180 ad, 1425 and 1450 ad, and 1520 and 1525 ad. Some of these changes are consistent with those inferred from new studies of the palaeoecological record of the Faroes. This indicates that the cumulative deviation measure could give greatest prominence to the most important climate changes affecting landscapes and settlement (such as the changes of 1425 and 1450 ad and their immediate aftermath), rather than extreme events, such as great single storms.     

Impact on disease development, genomic location and biological function of copy number alterations in non-small cell lung cancer

Lung cancer, of which more than 80% is non-small cell, is the leading cause of cancer-related death in the United States. Copy number alterations (CNAs) in lung cancer have been shown to be positionally clustered in certain genomic regions. However, it remains unclear whether genes with copy number changes are functionally clustered. Using a dense single nucleotide polymorphism array, we performed genome-wide copy number analyses of a large collection of non-small cell lung tumors (n = 301). We proposed a formal statistical test for CNAs between different groups (e.g., non-involved lung vs. tumors, early vs. late stage tumors). We also customized the gene set enrichment analysis (GSEA) algorithm to investigate the overrepresentation of genes with CNAs in predefined biological pathways and gene sets (i.e., functional clustering). We found that CNAs events increase substantially from germline, early stage to late stage tumor. In addition to genomic position, CNAs tend to occur away from the gene locations, especially in germline, non-involved tissue and early stage tumors. Such tendency decreases from germline to early stage and then to late stage tumors, suggesting a relaxation of selection during tumor progression. Furthermore, genes with CNAs in non-small cell lung tumors were enriched in certain gene sets and biological pathways that play crucial roles in oncogenesis and cancer progression, demonstrating the functional aspect of CNAs in the context of biological pathways that were overlooked previously. We conclude that CNAs increase with disease progression and CNAs are both positionally and functionally clustered. The potential functional capabilities acquired via CNAs may be sufficient for normal cells to transform into malignant cells.